MECHANISM OF LIPOPOLYSACCHARIDE-TRIGGERED JUNB ACTIVATION IN A MOUSE MACROPHAGE-LIKE CELL-LINE (J774)

The effect of lipopolysaccharide (LPS) on the activation of junB in a mouse macrophage cell line (J774) was investigated. J774 cells respond ed to either phorbol 12-myristate 13-acetate (PMA) or LPS by the trans ient increase in the expression levels of c-jun and junB mRNA, but not of junD mRNA. The prior depletion of protein kinase C from J774 cells blocked the action of PMA, but not of LPS, to activate junB. Pretreat ment of cells with H-89 or H-7, but not with HA1004, W-7, ML-7, or tyr phostin 47, inhibited LPS-triggered junB activation. Treatment with fo rskolin also activated junB of J774 cells through an H-89- or H-7-sens itive pathway. Since cAMP-dependent protein kinase activity of J774 ce lls was inhibited by H-89, but not by H-7, LPS appears to activate jun B through a cascade involving two steps, the one sensitive to H-89 and the other to H-7. Western blot analysis showed that LPS-triggered jun B activation is accompanied by the increased expression of JunB protei ns in the cell lysate as well as in the nuclear extract. JunB in nucle ar fraction appears to specifically bind to 12-O-tetradecanoylphorbol- 13-acetate-response element (TRE), since preincubation of nuclear extr acts with anti-JunB serum reduced the amount of TRE-binding proteins a nd since the amount of JunB, but not of c-Jun or JunD, immunoprecipita ted from TRE-cross-linked nuclear proteins increased in response to LP S. Thus, JunB may play an important role in LPS-triggered gene activat ion.